PROJECT SUMMARY/ABSTRACT Defining neuropathological features of Alzheimer?s disease (AD) include extracellular amyloid plaques and intracellular neurofibrillary tangles. The amyloid-? peptide (A?), the principal component of amyloid plaques, is derived from the serial proteolytic cleavage of the amyloid precursor protein (APP) by ?- and ?-secretase. Recent studies have demonstrated that ?-secretase, endocytosed via an independent pathway from APP, interacts with APP in early endosomes to produce ?-C-terminal fragment (?-CTF), which is trafficked down the canonical endolysosome pathway. The ?-secretase complex then cleaves ?-CTF in endosomes/multi-vesicular bodies (MVBs) to generate A?, some of which has been shown to be released with exosomes. Work over the last decade has demonstrated the importance of an alternate pathway in modulating A? production by diverting APP away from the endo-lysosome pathway back to Golgi via retromers?a heteropentameric protein complex which mediates retrograde transport of transmembrane proteins to the Golgi. SorLA (sorting protein-related receptor containing low-density receptor class A repeats), which binds to the retromer complex, also binds APP; and its disruption results in increased A? production. How retromer disruption alters APP processing and A? generation is unclear. Indeed, one report indicates that A? might even be generated in the Golgi. Some of these previous studies have been limited by the static nature of the cellular analysis?few probes are available to study in living cells. Ultimately, a dynamic quantitative approach to interrogate APP trafficking and de novo A? generation will be important for understanding how intracellular trafficking influences AD pathogenesis. In this grant, I propose to use a novel strategy to label A? within its precursor protein in living cells to track the intracellular sorting of APP and A? generation. I will use unnatural amino acid (UAA) mutagenesis and click chemistry, a labeling technique first applied to A?/APP by our lab, to site-specifically insert a small molecule fluorescent probe in the A? region within APP. APP, in turn, will be fused to a fluorescent protein in the C-terminus to create a bifluorescent APP construct; A? production will be indicated by separation of the two fluorescent probes. The trafficking and processing pathways of APP and its fragments will be investigated in both neural cell lines and primary neurons. The true spatial dynamics of APP processing and A? generation are most appreciated in the complex architecture of neurons, where APP is synthesized in the cell body and transported down dendrites and axons where the location and mechanisms of subsequent processing steps are unclear. This research will give insight into the underlying mechanisms of AD pathogenesis.